The present invention relates generally to an economical single channel method and system for detecting, identifying and quantifying different blood cell types, including platelets, in a mammalian blood sample, including human and non-human whole blood cell samples. Hemoglobin analysis is also provided by the method and system of the present invention. The method and system of the invention are particularly economical and useful for automated hematology analyzers utilizing flow cytometry systems.
The detection, identification and quantification of cellular and particulate blood components in a whole blood sample are necessary and customary parameters of blood sample analysis using hematology analyzers involving flow cytometry. A number of semi-automated and automated hematology analyzers can perform blood sample analyses; however, advancements in the technology of hematology analyzers and systems to afford further refinement, economy and accuracy to blood sample analysis advance and improve the ability to distinguish the various blood cell types and perform the necessary blood component analyses on whole blood samples.
Examples of automated hematology analyzers suitable for distinguishing and quantifying red blood cells, including mature red blood cells (RBCs) and reticulocytes, white blood cells (WBCs), including neutrophils, lymphocytes, monocytes, eosinophils and basophils; platelets; and hemoglobin include, but are not limited to the Bayer (formerly Technicon) H*(trademark) Systems series of hematology analyzers (including H*3(trademark) and H*Next(trademark) Systems) and the ADVIA(copyright) 120 Hematology System, which are developed and sold by the assignee hereof. The Bayer ADVIA(copyright) 120 System is a quantitative multi-channel, multi-dilution automated hematology analyzer that provides red blood cell and platelet analyses, as well as leukocyte (i.e., white blood cell) and reticulocyte analysis for in vitro diagnostic use in clinical laboratories.
Prior to the present invention, the use of hematology analyzers such as those provided above, required separate channels, reagents and channel detection optics for measuring, detecting and distinguishing among the mature red blood cells, reticulocytes, platelets and white blood cells, including neutrophils, lymphocytes and basophils, monocytes and eosinophils in a blood sample. For example, in previous methods and systems, one channel of a hematology analyzer is devoted to the analysis of red blood cells and platelets, a second channel is devoted to the analysis of reticulocytes; a third channel is devoted to the analysis of white blood cells; a fourth channel is devoted to basophil analysis; and a fifth channel is devoted to hemoglobin analysis. Correlated with the use of the different channels in an analyzer is the use of a number of different reagents, e.g., on the order of about five to eight distinct reagents, for diluting a number of aliquots of blood to elucidate the various cell types from one another.
In contrast to prior methods, the present invention provides a single dilution, single measurement channel method, e.g., approximately equivalent to the reticulocyte channel of an automated hematology analyzer and requiring only two reagents, i.e., an aqueous organic dye-containing reagent, preferably a cationic dye, and a sheath/rinse reagent, to discriminate among and measure the parameters of the various types of cells in the red and white blood cell groups, as well as platelets, in a mammalian whole blood sample, and to provide information on hemoglobin in the same sample. More specifically, the method of the present invention provides CBC/Diff/Retic determinations in a single measurement channel, including so-called reticulated platelet counts and percentages. The term xe2x80x9cCBCxe2x80x9d in the CBC/Diff/Retic determinations is defined as the complete blood count and includes determinations of the following: WBC (white blood cell count; 103/xcexcl), RBC (red blood cell count; 106/xcexcl), pLT (platelet count; 103/xcexcl), HGB (hemoglobin concentration; g/dl), HCT (hematocrit; %), MCV (mean cell volume; fl), MCH (mean cell hemoglobin; pg), MCHC (mean cell hemoglobin concentration; g/dl), RDW (red blood cell volume distribution width; %), HDW (cellular hemoglobin concentration distribution width; g/dl, which is a measure of the variability of cellular hemoglobin concentration within a sample), CHDW (distribution width associated with MCH, wherein (Vxc3x97hemoglobin concentration=cellular hemoglobin mass), MPV (mean platelet volume), MPC (mean platelet component concentration, g/dl), MPM (mean platelet dry mass), %Neutrophils, [%Lymphocytes+%Basophils], %Monocytes, %Eosinophils, Absolute Reticulocyte Count (109/l), %Reticulocytes, Reticulocyte MCV, Reticulocyte MCH and Reticulocyte MCHC, as well as absolute and percentage reticulated platelets.
The term xe2x80x9cDiffxe2x80x9d in the CBC/Diff/Retic determinations is defined as the white blood cell differential which includes determinations of % (percent) neutrophils, [%lymphocytes+%basophils], %monocytes and %eosinophils, as well as MNV (mean neutrophil volume), MNC (mean neutrophil component concentration, g/dl), MNM (mean neutrophil dry mass MLV (mean lymphocyte+basophil volume), MLC (mean lymphocyte+basophil component concentration, g/dl), MLM (mean lymphocyte+basophil dry mass MMV (mean monocyte volume), MMC (mean monocyte component concentration, g/dl), MMM (mean monocyte dry mass) MEV (mean eosinophil volume), MEC (mean eosinophil component concentration, g/dl), and MEM (mean eosinophil dry mass).
The term xe2x80x9cReticxe2x80x9d in the CBC/Diff/Retic determinations is defined as reticulocytes and includes the absolute reticulocyte count, including %Reticulocytes; Reticulocyte MCV; Reticulocyte MCH and Reticulocyte MCHC. It also includes Reticulated Platelets and the absolute reticulated platelet count and %reticulated platelets.
It is an object of the present invention to provide a method and system involving a single channel for the detection and measurement of the various types of red and white blood cells, including platelets, in a whole blood sample. Both human and non-human mammalian blood samples are analyzable by the present invention.
In accordance with the method of the present invention, at least three signals are collected and used to determine information about each cell that passes through the flow cell detector of a hematology analyzer substantially one cell at al time. The signals include two scatter signals and either one absorption signal (i.e., scatter/scatter/absorption) or one fluorescence signal (i.e., scatter/scatter/fluorescence). In its simplest aspect, only three signals are needed in the present single channel, single dilution method to count and distinguish from each other all of the platelets, red blood cell types and white blood cell types in a human or non-human mammalian blood sample, including a whole blood sample, based on either the scatter/scatter/absorption or the scatter/scatter/fluorescence pattern. Also according to the new single channel measurement method provided herein, a hematology analyzer may be designed, or adapted, so that it is physically streamlined in terms of space and volume to carry out the analysis of red and white blood cells and platelets, compared with prior analyzers that perform similar functions.
It is another object of the present invention to provide the following parameters and determinations of a complete blood count, i.e., a CBC/Diff/Retic, by the practice of the present single measurement channel method, wherein the CBC includes: WBC (white blood cell count; 103/xcexcl), RBC (red blood cell count; 106/xcexcl), PLT (platelet count; 103/xcexcl), HGB (hemoglobin concentration; g/dl), HCT (hematocrit; %), MCV (mean cell volume; fl), MCH (mean cell hemoglobin; pg), MCHC (mean cell hemoglobin concentration; g/dl), RDW (red blood cell volume distribution width; %), HDW (cellular hemoglobin concentration distribution width; g/dl), CHDW (distribution width associated with MCH, wherein (Vxc3x97hemoglobin concentration=cellular hemoglobin mass), MPV (mean platelet volume), MPC (mean platelet component concentration, g/dl), MPM (mean platelet dry mass). DIFF includes %Neutrophils, [%Lymphocytes+%Basophils], %Monocytes, %Eosinophils, MNV, MLV, MMV, MEV, MNC, MLC, MMC, MEC, MNM, MLM, MMM, MEM; Retic includes Absolute Reticulocyte Count (109/xcexcl), %Reticulocytes, Reticulocyte MCV, Reticulocyte MCH and Reticulocyte MCHC, as well as absolute reticulated platelet count and %reticulated platelets. HGB is computed as RBCxc3x97MCVxc3x97MCHC/1000.
It is another object of the present invention to provide a simpler and more convenient method and system for discriminating among the various types of red and white blood cells in a sample, because practice of the method requires only a single reaction chamber of an analyzer and a single dilution step to provide complete blood analysis parameters and values as enumerated above. In addition, the method comprises merely two reagents to achieve the CBC/Diff/Retic blood analysis parameters, namely, 1) a blood diluent reagent composition, preferably an xe2x80x9cautoreticxe2x80x9d or xe2x80x9creticxe2x80x9d reagent (e.g., Bayer ADVIA(copyright) Autoreticulocyte Reagent), comprising an organic dye compound, preferably, an organic cationic dye compound, for sample analysis, and 2) a sheath/rinse reagent (e.g., Bayer ADVIA(copyright) Universal Rinse Reagent as described in U.S. Pat. No. 5,888,742 to M. Malin et al., the contents of which are hereby incorporated herein by reference) to perform and achieve the detection and quantification results that are afforded by the invention. The term xe2x80x9cautoreticxe2x80x9d or xe2x80x9creticxe2x80x9d reagent as used herein is a shorthand nomenclature referring to a reagent previously used only in the reticulocyte channel of an automated hematology analyzer to identify, distinguish and measure only the red blood cell and reticulocyte populations in a blood sample. A significant advantage of the present single channel method of the present invention is that a single channel cycle is used in conjunction with a single dilution of the blood sample in the diluent reagent. Using only a single blood diluent mixed with a blood sample aliquot, the sample can be analyzed and a complete CBC, DIFF and reticulocyte analysis can be achieved.
Another object of the present invention is to provide a multispecies analysis method and system such that the blood cells of other species can be distinguished and analyzed using the single channel, single dilution method and system to gather and process three optical signals to distinguish white blood cells from all other blood cells, to perform CBC/DIFF/Retic analyses, including reticulated platelet analyses and white blood cell indices for other species.
Further objects and advantages afforded by the invention will be apparent from the detailed description hereinbelow.